Christopher A. O'Callaghan

HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C.

The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules,. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and β2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.

Functional Heterogeneity and High Frequencies of Cytomegalovirus-Specific CD8+ T Lymphocytes in Healthy Seropositive Donors

ABSTRACT Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8+ T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8+ T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in “fresh” killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E.

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Structure and function of the human MHC class Ib molecules HLA-E, HLA-F and HLA-G.

Summary: The major histocompatibility (MHC) class Ib molecules HLA‐E, HIA‐F and HLA‐G are relatively non‐polymorphic compared to class la molecules. Both HLA‐E and HLA‐G bind peptides and are involved in natural killer (NK)‐cell recognition, but the role of HLA‐F is unclear. HLA‐E binds specifically to the conserved leader sequence peptides from the class la MHC molecules and interacts on the cell surface with the CD94/NKG2 class of NK‐cell receptors. The framework structure of HLA‐E is similar to that of the MHC class la molecules, but the peptide‐binding groove is highly adapted for the specific binding of the leader sequence peptides. This is different from class la molecules, which have highly promiscuous peptide‐binding grooves. The HLA‐E groove makes full use of all the available pockets and imposes specificity along the entire length of the peptide. HLA‐G binds nonamer peptides with leucine or isoleucine at position 2, proline at position 3 and leucine at position 9. Expression of HLA‐G inhibits NK cells expressing the CD94/NKG2 class of receptors, though an interaction with these receptors has not been directly demonstrated.

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA‐F is a human non‐classical MHC molecule. Recombinant HLA‐F heavy chain was refolded with β2‐microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high‐affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA‐F. HLA‐F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA‐F could be immunoprecipitated from B cell lines and from HUT‐78, a T cell line. HLA‐F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA‐F tetramers stain peripheral blood monocytes and B cells. HLA‐F tetramer binding could be conferred on non‐binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA‐F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA‐F, suggest that HLA‐F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.

Differential Narrow Focusing of Immunodominant Human Immunodeficiency Virus Gag-Specific Cytotoxic T-Lymphocyte Responses in Infected African and Caucasoid Adults and Children

ABSTRACT Cytotoxic T-lymphocyte (CTL) activity plays a central role in control of viral replication and in determining outcome in cases of human immunodeficiency virus type 1 (HIV-1) infection. Incorporation of important CTL epitope sequences into candidate vaccines is, therefore, vital. Most CTL studies have focused upon small numbers of adult Caucasoid subjects infected with clade-B virus, whereas the global epidemic is most severe in sub-Saharan African populations and predominantly involves clade-C infection in both adults and children. In this study, sensitive enzyme-linked immunospot (elispot) assays have been utilized to identify the dominant Gag-specific CTL epitopes targeted by adults and children infected with clade-B or -C virus. Cohorts evaluated included 44 B-clade-infected Caucasoid American and African American adults and children and 37 C-clade-infected African adults and children from Durban, South Africa. The results show that 3 out of 46 peptides spanning p17Gag and p24Gagsequences tested contain two-thirds of the dominant Gag-specific epitopes, irrespective of the clade, ethnicity, or age group studied. However, there were distinctive differences between the dominant responses made by Caucasoids and Africans. Dominant responses in Caucasoids were more often within p17Gag peptide residues 16 to 30 (38 versus 12%; P < 0.01), while p24Gag peptide residues 41 to 60 contained the dominant Gag epitope more often in the African subjects tested (39 versus 4%;P < 0.005). Within this 20-mer p24Gag, an epitope presented by both B42 and B81 is defined which represents the dominant Gag response in >30% of the total infected population in Durban. This epitope is closely homologous with dominant HIV-2 and simian immunodeficiency virus Gag-specific CTL epitopes. The fine focusing of dominant CTL responses to these few regions of high immunogenicity is of significance to vaccine design.

Regulation of ligands for the activating receptor NKG2D

The outcome of an encounter between a cytotoxic cell and a potential target cell depends on the balance of signals from inhibitory and activating receptors. Natural Killer group 2D (NKG2D) has recently emerged as a major activating receptor on T lymphocytes and natural killer cells. In both humans and mice, multiple different genes encode ligands for NKG2D, and these ligands are non‐classical major histocompatibility complex class I molecules. The NKG2D–ligand interaction triggers an activating signal in the cell expressing NKG2D and this promotes cytotoxic lysis of the cell expressing the ligand. Most normal tissues do not express ligands for NKG2D, but ligand expression has been documented in tumour and virus‐infected cells, leading to lysis of these cells. Tight regulation of ligand expression is important. If there is inappropriate expression in normal tissues, this will favour autoimmune processes, whilst failure to up‐regulate the ligands in pathological conditions would favour cancer development or dissemination of intracellular infection.

Recognition and Blocking of Innate Immunity Cells by Candida albicans Chitin

ABSTRACT Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.

Molecular competition for NKG2D: H60 and RAE1 compete unequally for NKG2D with dominance of H60.

NKG2D is a potent activating receptor on natural killer cells, T cells, and macrophages. Mouse NKG2D interacts with two cell surface ligands related to class I MHC molecules: RAE1 and H60. We used soluble versions of NKG2D, RAE1, and H60 to characterize their interactions. RAE1 and H60 each bind NKG2D with nanomolar affinities, indicating tighter binding than most cell surface immune interactions, but NKG2D binds to H60 with approximately 25-fold higher affinity than to RAE1. RAE1 and H60 compete directly for occupancy of NKG2D, and, thus, NKG2D can be occupied by only one ligand at a time. The NKG2D-H60 interaction is more temperature dependent and makes greater use of electrostatic interactions than the NKG2D-RAE1 interaction. The distinct thermodynamic profiles provide insights into the different molecular mechanisms of the binding interactions.

CLEC-2 activates Syk through dimerization.

The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.